Methods of inhibiting smooth muscle cell proliferation and preventing restenosis with a vector expressing RB2/p130

ABSTRACT

Methods of inhibiting vascular smooth muscle cell proliferation and preventing restenosis by transducing the vascular smooth muscle cells with a viral veactor expressing RB2/p130 are provided.

This application is the United States national stage of InternationalApplication No. PCT/US99/20723, filed Sep. 10, 1999, which was publishedunder PCT Article 21 (2) in English as International Publication No. WO00/15649, and which claims benefit of priority of U.S. ProvisionalApplication No. 60/099,896 filed Sep. 11, 1998, now abandoned.

INTRODUCTION

This invention was made in the course of research sponsored by theNational Institutes of Health. The U.S. Government may have certainrights in this invention.

BACKGROUND OF THE INVENTION

Cardiovascular disease is the leading cause of mortality in the westernworld (Ross, R. Nature 362, 801-809 (1993); Landau et al. N. Engl. J.Med. 330, 981-993 (1994)) In the United States and other industrializedcountries ischemic heart disease, resulting in angina pectoris,myocardial infarction (MI), and sudden death, prevails as the principalcause of death and comprises at least 80% of all deaths from heartdisease (Braunwald, E. In Heart disease: a textbook of cardiovascularmedicine. Saunders W. B. Ed., (1996)) The vast majority of cases ofischemic heart disease are a consequence of atherosclerosis of thecoronary arteries (Ross, R Nature 362, 801-809 (1993)). The treatment ofocclusive coronary artery disease (CAD) involves three approaches, usedindividually or in combination: 1) coronary artery by-pass grafting(CABG); 2) percutaneous transluminal coronary angioplasty (PTCA), withor without stent application; and/or 3) medical management. While PTCAis less invasive and more cost effective than CABG, it bears somelimitations. One disadvantage of PTCA is that restenosis of the arteryoccurs in as many as 30 to 50% of cases within 3 to 6 months of theprocedure (Landau et al. N. Engl. J. Med. 330,981-993 (1994); Braunwald,E. In Heart disease: a textbook of cardiovascular medicine. Saunders W.B. Ed., (1996); Serruys et al. Circulation. 77,361-71 (1988); Holmes etal. Am. J. Cardiol. 53, 77C-81C (1984); Guiteras et al. Am. J. Cardiol.60, 50B-55B (1987)) . A variation of this technique is stent applicationin the dilated artery segment. Following promising preliminary reports,further investigation demonstrated that in the long-term the percentageof restenosis following stent-PTCA is similar to that of PTCA alone. Infact, in-stent restenosis is a frequent complication of stent-PTCA(Belli, G., Ellis, S. G., Topol, E. J. Stenting for ischemic heartdisease. In “progress in cardiovascular diseases” W. B. Sunders Ed. pp159-182 (1997)). No effective treatment for hindering restenosis iscurrently available. It is well documented that chronic or acute injury(such as from PTCA) to the arterial wall induces the expression of avariety of growth factors and inflammatory cytokines that stimulatesmooth muscle cell (SMC) proliferation and migration from the media intothe intirna resulting in neointima formation and eventual restenosis(Clowes et al. Lab. Invest. 49, 208-215 (1983)). Inhibition of neointimaformation should greatly improve the effectiveness of PTCA in the longterm management of CAD. Numerous growth factors induce SMC proliferationthrough a variety of signal transduction pathways in vitro and in vivo(Libby et al. Circulation 86 (Suppl III) 47-52 (1992)). Accordingly,several regulatory proteins of the cell cycle machinery, instead of theupstream signal transduction molecules, have been suggested as targetsfor effective cytostatic therapy of vascular proliferative disorders.For example, Morishita et al. disclose studies wherein singleintraluminal delivery of antisense cdc2 kinase and proliferating-cellnuclear antigen oligonucleotides resulted in chronic inhibition ofneointimal hyperplasia. Proc. Acad. Natl. Sci. 90, 8474-8478 (1993).Chang et al. disclose results from studies suggested to demonstrate therole of (retinoblastoma) Rb in regulating vascular smooth muscle cellproliferation and to suggest a gene therapy approach for vascularproliferation disorders associated with arterial injury. Science. 267,518-522 (1995). Smith et al. also analyzed the effects of full-lengthphosphorylation competent and mutant truncated forms of human Rb fortheir effects on vascular smooth muscle proliferation and neointimaformation. Circulation 1997 96(6) 1717-9. These analyses are taught toreveal that the maintenance of high levels of phosphorylation competenthuman Rb either full-length or truncated forms in vascular smooth musclecells is an effective method of modulating the extent of intimalhyperplasia that occurs after balloon induced vascular injury. Smith etal. Circulation. 96, 1899-1905 (1997)).

Another member of the retinoblastoma family, RB2/p130, has also beenshown to have a regulatory role in cell cycle function. Baldi et al.have shown that phosphorylation of the RB2/pl30 gene product isregulated in a cell cycle dependent manner (Baldi et al. J. Cell.Biochem. 59:402-408 (1995)), in the same way that the phosphorylation ofRb is cell cycle dependent (DeCaprio et al. Cell 58:1085-1095 (1989)).Further, the growth suppressive properties of the gene product ofRB2/pl30 have been shown to be specific for the G₁ phase in similarfashion to pRb and pl07 (Claudio et al. Cancer Res. 56:2003-2008(1996)). The gene product of RB2/pl30 has also been shown to arrestgrowth in human tumor cell lines in a manner similar to the othermembers of the Rb family (i.e., pRb and pl07). However, this proteinalso inhibits proliferation in a glioblastoma cell line that isresistant to the growth suppressive effects of both pRb and pl07(Claudio et al. Cancer Res. 54:5556-5560 (1994)). Accordingly, RB2/pl30has similar yet distinctive growth suppressive properties from pRb andpl07 (Claudio et al., Cancer Res. 54:5556-5560 (1994)).

It has now been found that localized arterial transduction of RB2/pl30via a viral vector at the time of angioplasty drastically reducesneointimal hyperplasia and prevents restenosis. Furthermore, the abilityof RB2/pl30 to block proliferation correlates with its ability to bindand sequester the E2F family of transcription factors, which areimportant mediators of cell cycle progression. Accordingly, RB2/pl30 isbelieved to be an important target for vascular gene therapy.

SUMMARY OF THE INVENTION

An object of the present invention is to provide a method of inhibitingvascular smooth muscle cell proliferation which comprises transducingvascular smooth muscle cells with a viral vector expressing RB2/pl30.

Another object of the present invention is to provide a method ofpreventing restenosis in a patient which comprises administering to apatient a viral vector expressing RB2/pl30.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1a-1 b show the effects of adenoviral transduction of the RB2/pl30gene on the growth of rat pulmonary artery smooth muscle cells (PASM)and Western blot demonstrating pRb2/pl30 expression in PASM cellstransduced with Ad-CMV (empty viral vector) or Ad-CMV-Rb2/pl30 overtime.

FIGS. 2a-2 c show the effects of increasing quantities of Adenoviralparticles on the growth of a pulmonary artery smooth muscle cell line(PASM).

FIGS. 3a-3 b show the effects of pRb2/pl30 induction on E2F-bindingcapacity.

FIGS. 4a-4 b is the graphic representation of lumen size (A) andarterial wall thickness (B) post angioplasty in Ad-CMV, Ad-RB2/pl30transduced carotid arteries and left carotid arteries (control).

FIG. 5 shows structural and ultrastructural analysis of CMV and Rb2vessels.

FIG. 6 is the Western blot analysis of E2F-4 and 5 in PASM cellstransduced with either Ad-CMV or Ad-RB2/pl30.

FIG. 7 shows structural and ultrastructural analysis of control vessels.

FIG. 8 shows immunohistochemical analysis of pRb2/pl30 andβ-galactosidase in uninjured, Ad-RB2/pl30 and β-galactosidase transducedcarotid arteries.

DETAILED DESCRIPTION OF THE INVENTION

Smooth muscle cell (SMC) proliferation resulting in neointima formationis implicated in the pathogenesis of atherosclerotic plaques andaccounts for the high rates of restenosis following percutaneoustransluminal coronary angioplasty (PTCA) with or without stentapplication, a widespread treatment for coronary artery disease (CAD).Endothelial lesions trigger intense proliferative signals to the SMCs ofthe subintima, stimulating their re-entry into the cell cycle from aresting G₀ state, resulting in neointima formation and vascularocclusion. Cellular proliferation is negatively controlled by growthregulatory and/or tumor suppressor genes, such as the retinoblastoma(Rb) gene family members (RB/pl05, pl07, RB2/pl30).

Accordingly, the retinoblastoma family proteins (pRb/pl05, pl07, andRB2/pl30) are excellent candidates for vascular disease gene therapy.They are nuclear phosphoproteins with growth suppressive properties thatinteract with specific members of the E2F transcription factor family(E2F1-5) and are regulated by phosphorylation/dephosphorylation eventsin a cell cycle dependent manner (Weinberg, R. A. Cell 85, 457-459(1996); and Nevins et al. J. Cell. Physiol. 173, 233-236 (1997)).Studies have shown that induction of RB2/pl30 expression growth arrestsproliferating cells in the G₀/G₁ phase of the cell cycle by directlyinteracting with and regulating the activity of the cell cycle machinery(Claudio et al. Cancer Research 54, 5556-5560 (1994); Claudio et al.Cancer Res. 56, 2003-2008 (1996); and De Luca et al. J. Biol. Chem. 272,20971-20974 (1997)) Furthermore, induction of pRB2/pl30 expressioninhibits cellular proliferation in certain cell lines that arerefractory to the effects of Rb family members pRb/pl05 and pl07(Claudio et al. Cancer Research 54, 5556-5560 (1994); De Luca et al. J.Biol. Chem. 272, 20971-20974 (1997)). It has now been found thatadenovirus-mediated transduction of RB2/pl30 blocks SMC proliferation invivo and in vitro. Moreover, it has now been shown that RB2/pl30preferentially interacts with and sequesters the growth promotingtranscriptional activity of E2F4 in SMCs.

Adenoviruses have been shown to serve as effective and efficient vectorsto deliver transgenes to cells in vivo. Accordingly, in theseexperiments, Adenoviral mediated-RB2/pl30 gene transfer was used to holdin check the proliferative capacity of the arteriai smooth musclesubsequent to acute injury in order to prevent restenosis followingangioplasty.

The effects of viral transduction of the RB2/pl30 gene on the growth ofa pulmonary artery smooth muscle cell line (PASM) was examined. The PASMcell line expresses many differentiation markers off SMCs and istherefore considered a good paradigm (Roberts et al. EMBO J. 15,6301-6310 (1996); Rothman et al. Circulation. 86, 1977-1986 (1992)). Inthese experiments, PASM cells were plated at a density of 1×10⁵ in 10 cmdiameter dishes in triplicates and transduced with 50 pfu/cell of eitherAd-CMV or Ad-CMV-RB2/pl30. Cells were counted by trypan blue exclusionmethod each day over a week to monitor their growth rate. In PASM cellstransduced with Ad-CMV-RB2/pl30 the growth rate was inhibited by 5 foldas compared to that of Ad-CMV transduced cells.

To measure the expression level of RB2/pl30 in Ad-CMV-RB2/pl30 andAd-CMV transduced cells, cells were harvested each day for one week andwestern blot analysis was performed on extracts. At each time point, theexpression of RB2/pl30 was ˜200 fold higher in the Ad-CMV-RB2/pl30 thanin the Ad-CMV transduced cells. The blot was normalized for equalloading and transfer of proteins by blotting the membrane with anti-βTubulin as well as staining the membrane with Coomassie brilliant blue.

The effects of different amounts of Adenoviral particles on the growthof a pulmonary artery smooth muscle cell line (PASM) was also examined.In this experiment, PASM cells were plated at a density of ×10⁵ in 10 cmdiameter dishes in triplicates and transduced with 5, 10 or 50 pfu/cellof either Ad-CMV, Ad-CMV-β-Gal or Ad-CMV-Rb2/l30. The cells were countedby trypan blue exclusion method each day over 5 days to monitor theirgrowth rate. In PASM cells transduced with 10 or 50 MOI ofAd-CMV-Rb2/pl30 the growth rate was inhibited by almost 4 fold ascompared to that of Ad-CMV and Ad-CMV-β-Gal transduced cells. Cell countin Ad-CMV infected PASM cells was found comparable to that ofAd-CMV-β-Gal infected cells. To test whether exposure of the culturedcells to Adenovirus at 50 MOI could be associated to toxicity PASM cellswee transduced with 50 MOI of either Ad-CMV, Ad-CMV-β-Gal orAd-CMV-RB2/pl30 and 48 hours later processed the samples for Facsanalysis. It was observed that exposure of PASM cells to any of thethree adenoviruses did not cause apparent cytotoxicity. Further, theoverexpression of RB2/pl30 resulted in a G₁₀ accumulation of the cellscompared to the control (Ad-CMV and Ad-CMV-β-Gal) transduced cells.

RB2/pl30 was also demonstrated to associate in vivo with E2F familymembers and to inhibit their ability to transactivate genes that promotecell cycle in PASM cells. In vivo RB2/pl30 associates with E2F4 and E2F5and thereby inhibits their ability to transactivate genes that promotecell cycle progression (Sardet et al. Proc. Natl. Acad. Sci. USA 92:2403-2407 (1995)). Since the growth suppressive function of theretinoblastoma family of proteins is thought to occur at least in partby their binding and negative regulation of specific members of the E2Ffamily of transcription factors, the E2F complexes in PASM cellsinfected with either Ad-CMV-Rb2/pl30 or Ad-CMV cells were analyzed. Inthese experiments, PASM cells were plated at a density of 2×10⁶ cellsand transduced with either the adenoviruses Ad-CMV or Ad-CMV-RB2/pl30 ata concentration of 50 pfu/cell and harvested after 48 hours. Byelectrophoretic mobility shift assay (EMSA) using a oligonucleotideprobe of the E2F DNA binding sequence labeled with [³²P]-γ dCTP, an E2Fcomplex was detected that effectively competed with cold wild-typeoligonucleotide but not with a point mutated oligonucleotide thatabrogates E2F binding to DNA. The band of the E2F complex wassupershifted by incubation with an antibody that specifically recognizesRB2/pl30 as well as by an antibody that specifically recognizes E2F4 inboth the Ad-CMV-RB2/pl30 and Ad-CMV transduced cells. Almost the entireE2F complex was shifted in the Ad-CMV-RB2/pl30 infected cells byincubation of the RB2/pl30 antibody, indicating that most of the E2F isbound by RB2/pl30 in these cells. However in the Ad-CMV transduced cellsonly a small fraction of the E2F complex was supershifted by theRB2/pl30 antibody, a reflection of the low endogenous expression levelof RB2/pl30 in the proliferating PASM cells. This indicates thattransduction of smooth muscle cells by Ad-CMV-RB2/pl30 and the resultinghigh level of expression of RB2/pl30 provides an abundance of RB2/pl30that can effectively sequester E2F activity thereby leading to growtharrest. Additionally, the band of the E2F complex, was supershifted byincubation with an antibody that recognize E2F4.

Expression of the E2F family members following transduction was alsoexamined. It was found that the expression level of E2F5 was noteffected by either adenovirus carrying RB2/pl30 or adenovirus control.The expression of E2F4 was instead down-regulated upon overexpression ofRB2/pl30, indicating that its overexpression in PASM cells targeted E2F4for protein instability and degradation or inhibited its transcription.

The vast majority of endogenous E2F activity during the G₀/G₁ transitionstems from E2F4 (Cobrinik et al. Genes & Development. 7, 2392-2404(1993)). In fact, during the G₁ phase of the cell cycle, E2F4 accountsfor almost all of the free activity. During S phase an equal mixture ofE2F4 and E2F1 comprises the free E2F activity (Moberg et al. Mol. &Cell. Biol. 16, 1436-1449 (1996)). The main form of E2F detected in theG₀-G₁ phases in primary mouse fibroblasts is E2F4 bound to RB2/pl30which is then replaced by pl07E2F4 complexes in late G₁ (Cobrinik et al.Genes & Development. 7, 2392-2404 (1993); Moberg et al. Mol. & Cell.Biol. 16, 1436-1449 (1996)). Thus, RB2/pl30 is a primary negativemodulator of E2F activity in quiescent or resting cells and offers thepotential for inhibiting cellular proliferation and intimal thickeningafter balloon angioplasty.

The effects of RB2/pl30 transduction on neointimal formation two weeksafter balloon injury were analyzed in vivo in a rat carotid artery modelof restenosis (Indolfi et al. Nature Med. 3, 775-779 (1997); Indolfi etal. Nature Med. 1, 541-545 (1995)). in the animal group infused withAd-CMV, a reproducible neointimal formation as shown by semithinsections stained with methylene blue was observed. An increase of theintimal thickness for the presence of several layers of smooth musclecells with the interruption of the inner elastic lamina was found. Thisresulted in a great decrease of the lumen (Lu) (276.4 μm±25.4), andconversely an increase in the thickness of the medial and intimalcarotid layer (Th) (308 μm±74) compared to the left carotid artery(Lu=758.8 μm±41.85, Th=57.4 μm±1.2) By electron microscopy the neointimaof the rats treated with Ad-CMV appeared almost entirely constituted oflayers of smooth muscle cells immersed into an intercellular matrix thatincluded reticular fibrils, without any trace of neovascularization.

In the group treated with Ad-RB2/pl30 neointimal formation was highlysuppressed 14 days after injury. The lumen of the right carotid arteriesperfused with Ad-RB2/pl30 (Lu=712.38 μm±117.36) was comparable to theleft uninjured side (Lu=652.63 μm±87.23). There were essentially nodifferences between the thickness of the arterial walls treated withAd-RB2/pl30 (Th=60.5 μm±6.18) and the normal uninjured left side(Th=64.8 μm±8.68). The differences between the animal group treated witheither Ad-CMV or Ad-RB2/pl30 were found to be statistically significant(P<.01). However, no statistically significant differences in the lumensize or in the arterial thickness were found between the right carotidartery treated with Ad-RB2/pl30 or the left untreated carotid arteryindicating that RB2/pl30 greatly inhibited the proliferative capacity ofthe smooth muscle cells in vivo as in vitro. By electron microscopy itwas demonstrated that the intimal layer of the carotid arteries treatedwith Ad-RB2/pl30 consisted solely of endothelial cells. Themorphological aspects of the Ad-RB2/pl30 treated carotid arteries werecompared with the contralateral uninjured side. Cross semithin sectionsof the left carotid arteries (controls) stained with methylene blue andobserved by light microscopy showed a normal thickness of the arterialwall as well as typical endothelial cells limiting the lumen asdetermined by electron microscopy identical to the Ad-RB2/pl30 treatedcarotid arteries.

The expression levels of RB2/pl30 post transduction in vivo were alsoexamined via imnmunohistochemistry. Sections of carotid arteries treatedwith Ad-RB2/pl30 showed a high level of RB2/pl30 when compared to theleft normal uninjured side, demonstrating in vivo adenoviral genetransfer of RB2/pl30 and the constitutively high expression of thetransduced gene after 2 weeks. The efficacy of gene transfer wasdetermined by identifying single cells positive for nuclear pRb2/pl30staining as well as by the high expression level of β-Galactosidasetransgene in Ad β-Gal infected vessels.

Accordingly, the present invention provides methods for inhibitingvascular SMC proliferation and for preventing restenosis in patient witha viral vector comprising RB2/pl30. In a preferred embodiment, RB2/pl30cDNA is subcloned into an adenoviral vector in accordance with methodsof plasmid construction well known in the art. As will be obvious tothose of skill in the art upon this disclosure, however, appropriatevectors other than those described herein. For example, in oneembodiment, a transient three-plasmid expression system (plasmidsencoding env, gag-pol, and RB2/pl30) can be prepared. This embodimentwould involve using a retroviral vector wherein the full length cDNAsequence of RB2/pl30 is subcloned into the vector. Selection of anappropriate vector is based upon adequate expression of the gene withminimal viral gene expression.

The viral vector encoding RB2/pl30 is then transduced into vascularsmooth muscle cells by contacting the cells with the vector. This can beaccomplished using methods known to those of skill in the art. In apreferred embodiment, the viral vectors are administered to a mammal,preferably a human. In one embodiment, the vector containing theRB2/pl30 gene is administered to suppress vascular smooth cellproliferation and prevent restenosis through expression of this cellproliferation suppressor gene. In this embodiment, the method oftransduction will preferably be performed by intra-arterial injection toarteries. To prevent restenosis following angioplasty, it is preferredthat the viral vector expressing RB2/pl30 be administered to the patientat the time of the procedure.

It is preferred that the viral vectors be administered in apharmaceutically acceptable carrier for injection such as a sterileaqueous solution or dispersion. Dose and duration of treatment isdetermined individually depending on the degree and rate of improvement.Such determinations are performed routinely by those of skill in theart.

The following nonlimiting examples are provided to further illustratethe present invention.

EXAMPLES Example 1 Plasmids

The full length ORF of the RB2/pl30 gene has been subcloned into thepAd-CMVlink-1vector by releasing the RB2/pl30 gene from thepCDNA3-RB2/pl30 construct with a HindIII-Sal I digestion and insertingit into the pAd-CMVLink-1 digested with HindIII (Claudio et al. CancerResearch 54, 5556-5560 (1994); Davis, A. R. and Wilson, J. M. AdenovirusVectors. Dracopoli et al. Eds. In Current Protocols in Human Genetics 2,Unit 12.4 (1996)). The construct pAd-CMVLink-1-βgal has been describedpreviously (Davis, A. R. and Wilson, J. M. Adenovirus Vectors. Dracopoliet al. Eds. In Current Protocols in Human Genetics 2, Unit 12.4 (1996)).The vector pAd-CMVLink-1 contains the Adenovirus 5 sequence encoding mapunits 0-1, a cytomegalovirus immediate early enhancer/promoter element,SV40 splice donor/acceptor site intron, SV40 polyadenylation signal,Adenovirus 5 sequence encoding map units 9-16, a synthetic multiplecloning site, and sequences from the pAT153 plasmid.

Example 2 Cell Lines

293 cells (primary embryonal human kidney cells) and PASM (PulmonaryArtery Smooth Muscle) cells were maintained in D-MEM containing 10%Fetal Bovine Serum (FBS) and L-Glutamine.

Example 3 Adenoviral Production and Transduction

The Ad-CMV-RB2/pl30, Ad-CMV and Ad-CMV-βgal were generated as follows.Recombinant adenoviruses were constructed by cotransfecting theadenoviral shuttle plasmid (pAd-CMVlink-1 with the insertion of eitherthe RB2/pl30 or the Lac-Z gene behind the CMV promoter) and Cla Idigested AdS DNA (“viral backbone”) containing a GFP selection marker,into 293 primary embryonal human kidney cells. The 293 cells serve as apackaging cell line that supplies the E1 function in trans. Homologousrecombination occurs in the region of overlap, in region E1 between therestricted Ad5 viral DNA (“viral backbone”) and the shuttle plasmid(pAdCMVLink-1-RB2/pl30). After transfection, 293 cells were overlaidwith agar-containing medium and after approximately 10 days visibleplaques were examined using an inverted fluorescent microscope.Non-fluorescent or “white” plaques were easily distinguished frombackground or “green” plaques. The white plaques were expanded byinfection of 293 cells and after appearance of complete cytopathiceffect, DNA was prepared by a modification of the method of Hirt (Arad,U. Biotechniques 24, 761-762 (1998)). DNA recombinant adenoviruses werecontrolled by restriction enzyme analysis. Recombinant adenoviruses werethen screened by western blot analysis for expression levels of RB2/pl30using an antibody that specifically recognizes RB2/pl30 Baldi et al. J.Cell. Biochem. 59, 402408 (1995)). The recombinant adenovirus thatshowed the highest expression of RB2/pl30 was plaque purified once moreand expanded by infection of 293 cells. Purification of the adenovirusesin the supernatant was performed by sequential equilibrium densitygradients using CsCl (Davis, A. R. and Wilson, J. M. Adenovirus Vectors.Dracopoli et al. Eds. In Current Protocols in Human Genetics 2, Unit12.4 (1996)). Purified viruses were stored in a solution containing 10%glycerol at −80° C. Optical density was measured and viral stocks weremade at 5×10¹² particles/ml. A viral titer of 22×10⁹ pfu/ml wasdetermined by plaque assay for the Ad-CMV and Ad-CMV-RB2/pl30 viruses. Aviral titer of 40×10⁹ pfu/ml was determined for the Ad-β-gal virus.Infection of non-permissive cells (cells lacking E1 function) confirmedthat the viruses were only able to undergo one round of infection;therefore, they are replication defective.

Example 4 Determination of Growth Rates and Western Blot

PASM cells were plated at a density of 1×10⁵/dish into 10 cm culturedishes in triplicate. Cells were transduced with 4.4×10⁷ pfu of eitherAd-CMV or Ad-CMV-RB2/pl30 in DMEM medium containing 2% Fetal BovineSerum (FBS). The next morning the medium was removed and replaced withDMEM containing 10% FBS. The cells were harvested every 24-hours for 6days and counted by trypan blue exclusion. Cells were also spun down andpellets were lysed. Western blot analysis was performed essentially aspreviously described (Claudio, et al. Cancer Res. 56, 2003-2008 (1996)).The anti-RB2/pl30 was used at a dilution of 1:1000, the anti-β-tubulin(Sigma, St. Louis, Mo.) was used at a dilution of 1:1000, and the E2F4and 5 antibodies (Santa Cruz, Calif.) were used at a dilution of 1:500.

Example 5 Dose-response Growth Curve and Facs Analysis

PASM cells were plated at a density of 1×10⁵/dish into 10 cm culturedishes in triplicate. Cells were transduced with 5, 10 or 50 MOI ofeither Ad-CMV, Ad-CMV-βGal or Ad-CMV-Rb2/pl30 in DMEM medium containing2% Fetal Bovine Serum (FBS). The next morning the medium was removed andreplaced with DMEM containing 10% FBS. The cells were harvested every 24hours for 5 days and counted by trypan blue exclusion for dose-responsegrowth curve. For Facs analysis purpose cells were collected 48 hoursafter transduction and fixed in 70% ethanol and treated with a solutioncontaining propidium iodide and RNAse A as previously described byClaudio et al., Cancer Research 56:2003-2008 (1996).

Example 6 Gel Shift Assay (EMSA)

PASM cells were seeded at a density of 1×10⁶/dish into 10 cm culturedishes in duplicate. Cells were transduced with 1×10⁸, pfu of eitherAd-CMV or Ad-CMV-RB2/pl30 in DMEM medium containing 2% FBS. The nextmorning the medium was removed and replaced with DMEM containing 10%FBS. Cells were collected and gel shift assays were performedessentially as previously described by Pagano et al. Science 255,1144-1147 (1992).

Example 7 Animal Studies

All WKY rats were maintained under identical conditions of temperature(21±1° C.), humidity (60±5%) and light/dark cycle, and had free accessto normal rat diet. The animal group treated with either Ad-CMV orAd-RB2/pl30 consisted of 10 rats each, while the group infused withAd-β-Gal consisted of 5 rats.

The right common artery of 35 WKY rats at 14 weeks of age (CharlesRiver, Morini, Italy) weighing 350 to 400 g was denuded of endotheliumby the intraluminal passage of a balloon embolectomy catheter (2F′Fogarty, Edwards Laboratories) introduced through the external carotidartery. All animals were anesthetized with an intraperitoneal injectionof ketamine (Ketalar, Parke-Davis) 80 mg/kg and xilazine (Rompun, BayerAG) 5 mg/kg. The right common and external carotid artery was surgicallyexposed and an arteriotomy was performed in the external carotid artery.Deendothelializing injury of a right common artery segment was achievedby insertion and passage of a 2F Fogarty balloon catheter, distendedsufficiently with saline solution to generate slight resistance by aninflation at 1.5 atm with a calibrated device (Indeflator Plus 20,Advanced Cardiovascular System, Inc.). To ensure adequate endothelialdamage, the inflated catheter was passed a total of three times. Thistechnique produced distension of the carotid itself. The time of ballooninflation was kept constant for twenty seconds. After balloon injury asegment of common artery approximately one cm in length was isolatedplacing vascular clamps on the proximal common artery and proximalinternal artery. A polyethylene catheter (PE 50, Hecton Dickinson) wasintroduced through the external carotid arteriotomy and the isolatedvessel segment was flushed with M-199 medium before introduction ofeither adenoviral vectors. Adenoviral vectors were defrosted, diluted(1×10⁹ pfu/μL) and kept in wet ice until use. All stocks were usedwithin two hours of thawing. For each rat a total of 50 μL dilutedadenoviral vector were instilled into the isolated common carotidsegment by means of a catheter placed in the external carotid artery.During infusion of the vector-containing medium, the isolated carotidsegment became distended and remained so for the duration of theinstillation period. After twenty minutes of incubation, thevector-containing medium was withdrawn, the external carotid artery wasligated and the blood flow through the common and internal carotidarteries was reestablished. Rats were allowed to recover from anesthesiaand were returned to their cages.

Example 8 Morphology

Two weeks after gene transduction, carotid arteries were dissected freefrom the surrounding tissues and rats were sacrificed.

Structural and ultrastructural analysis of blood vessel wall and lumenwere performed on specimens fixed in universal solution (4%paraformaldehyde and 1% glutaraldehyde in 0.1 M phosphate buffer). Thedelay to fixation was approximately 2-3 hours. Blood vessels were washedtwice in PBS and postfixed in 1% osmium tetroxide. Then the specimenswere rinsed two times in PBS, followed by dehydration through a seriesof alcohols (30% to 100%, 15 min steps). The embedding was performed inepoxy resin (Epon 812) and polymerized at 60° C. overnight. Semithincross sections (0.5 μm), were cut in a Ultracut-E (Reichert-Young),mounted onto slides and stained by warm methylene blue. Screening andphotography were performed using a Zeiss microscopy.

Ultrathin cross sections (60 nm) were mounted on copper grids,counterstained with uranyl acetate for 6 minutes and lead citrate for 5minutes. The specimens were analyzed by a JEOL JEM-1220 electronmicroscope.

Blood vessel wall thickness and lumen diameter were evaluated using aDigital Imaging Processing (Image-Pro Plus, Media Cybernetics, SilverSpring Md., USA). For immunohistochemical purposes, serial frozen bloodvessel sections (5 μm) were allowed to equilibrate to room temperaturefor 12 hours and exposed to acetone for 10 minutes. Abiotin-streptavidin (Vectastain Universal Quick Kit, VectorLaboratories, Inc., Burlingame, Calif.) preformed complex was used todetect the expression of RB2/pl30. Sections were preblocked with 10%horse serum/PBS+0.2% Tween-20 for 20 minutes at room temperature. Tissuesections were eliminated of excess serum and incubated with our specificpolyclonal anti-pRb2/pl30 or horse serum at appropriate dilutions (DeLuca et al. Mature Med. 3, 913-916 (1997)). After washing, bound primaryantibodies were detected by a universal biotinylated antibody predilutedin TBS at room temperature for 20 minutes followed by incubation for 10minutes with a peroxidase-conjugated streptavidin.3-amino-9ethylcarbazole (AEC) was used as the calorimetric substrate.Analysis of blood vessel sections was performed by a Zeiss lightmicroscopy.

Example 9 Statistical Analysis

All data shown are means ± s.e.m. Statistical analysis between groupswas performed by analysis of variance (ANOVA) using a Systat program(Systat INC, Evanston, Ill.). Tukey's test was applied to compare singlemean values when a significant overall effect was determined. Dixon, W.J. et al., Introduction to Statistical Analysis, New York, McGraw-Hill(1969). A P value of <.05 was considered statistically significant

All publications and references, including but not limited to patentapplications, cited in this specification, are herein incorporated byreference in their entirety as if each individual publication orreference were specifically and individually indicated to beincorporated by reference herein as being fully set forth.

While this invention has been described with a reference to specificembodiments, it will be obvious to those of ordinary skill in the artthat variations in these methods and compositions may be used and thatit is intended that the invention may be practiced otherwise than asspecifically described herein. Accordingly, this invention includes allmodifications encompassed within the spirit and scope of the inventionas defined by the claims.

What is claimed is:
 1. A method of inhibiting vascular smooth musclecell proliferation comprising transducing vascular smooth muscle cellsby intraarterial local instillation of an adenoviral vector expressingRB2/pl30.
 2. A method of preventing restenosis in a patient whichcomprises administering to a patient by intraarterial local installationof an adenoviral vector expressing RB2/pl30.